New intracellular biosensor
The RNA sensor began to be used to evaluate gene expression in a living cell
American bioengineers have created and tested an intracellular gene expression sensor based on a double-stranded RNA editing system. The design makes it possible to detect the desired RNA in the cytoplasm of a living cell without resorting to genomic editing and without disrupting gene expression. This technology, called CellREADR, can be adapted after simple modifications for optogenetic experiments and calcium imaging. The study is published in Nature (Qian et al., Programmable RNA sensing for cell monitoring and manipulation).
Studying the response of cells to drugs and the study of brain development often requires understanding how the transcription of individual cells changes. Modern transcriptomic technologies make it possible to analyze gene expression in single cells (and sometimes even without killing the cell), but they mainly concern ex vivo studies.
For situations where it is necessary to evaluate the change in gene expression in a living organism, there are genetic lines in which the reporter gene is regulated by the same sequences as the "gene of interest". But not for every protein and not for every model object there is a genetic line with an expression sensor built into the genome. Creating models is often long and difficult, and even modern genomic editing technologies are not without errors.
Bioengineers and neuroscientists from Duke University and the Cold Spring Harbor Laboratory, led by Josh Huang, have developed an RNA sensor for gene expression. CellREADR (Cell access through RNA sensing by Endogenous ADAR) technology is based on an intracellular system of post-transcriptional modification of RNA based on enzymes RNA-specific adenosine deaminases (ADAR). These enzymes, which are present in most animal cells, "search" for non-complementary pairs of nitrogenous bases "adenine-cytosine" in double-stranded RNAs in the cytoplasm. When such a pair is detected, ADAR triggers a chain of reactions, as a result of which adenine is replaced by guanine.
The proposed single-stranded RNA molecule (the authors called it readr-RNA) functionally consists of two parts. The sensory segment is 200-350 nucleotides long, 95-98 percent complementary to the desired RNA. It starts with a sequence that triggers translation, and ends with a stop codon that is partially complementary to the desired mRNA.
If such an RNA enters a cell in which there is no target RNA, then a non-functional protein is translated from it, and everything ends at the stop codon. But if the sensor binds to the target, then ADARs detect a violation of complementarity in the stop codon of readr-RNA. After activation of ADAR, the stop codon changes to the coding one, and the protein encoded in the second half of the RNA is translated. It encodes the protein T2A, capable of autoproteolysis, and a reporter protein (fluorescent, chemo, photosensor, or even an inducer of apoptosis). The synthesized RNA can be "packed" into a virus or into a liposome.
Having developed the concept, Dr. Huang and colleagues tested the technique on human cell culture. The experiment showed that false triggering occurs in 0.3-0.5 percent of culture cells, and sensitivity ranges from 10-50 percent, depending on gene expression. Experiments with changing the sensory sequence have shown that in one readr-RNA it is possible to connect two sensors to two different targets.
Having worked out the technology on cell cultures, the researchers tested it on multicellular organisms. They targeted readr-RNA to proteins specific to different sensory and motor subpopulations of neurons in the cerebral cortex of mice, rats and humans (in the latter case, scientists used live biopsies of brain tissue). The specificity ranged from 62-92 percent depending on the target protein and the population of neurons. If we used a signal sequence to two different RNAs, the specificity increased to 85-97 percent.
The specificity of CellREADR in identifying pyramidal neurons of the motor cortex. Figures from the article by Qian et al.
But you can use CellREADR not only to monitor activity, but also to manipulate it. In one of the experiments, Dr. Huang and colleagues added channelorhodopsin RNA (a protein that provides a response to light in unicellular algae) to the reporter part of readr-RNA aimed at the "motor" pyramidal neurons of the cortex of mice. Optogenetic stimulation of the animal's brain caused the movement of the paw. The speed and amplitude of this movement were the same as in "normal" optogenetics, when the rhodopsin gene is integrated into the genome.
The RNA sensor can also be used for optogenetic studies. The mouse moves its paw in response to photostimulation of the bark.
An experiment with the somatosensory cortex and the calcium-dependent protein GCaMP6 showed that READR can also be used for calcium visualization of neuronal activity. A safety study of readr-RNA showed that the level of neuroinflammation three months after transfection did not change, as did the expression of the target genes.
Based on the experiments conducted, the authors conclude that the technique is quite versatile and easily adaptable to different tasks. The new method works in vivo, does not depend on translation and on the role of the final protein product. It can be configured for different stages of post-transcriptional modification of RNA, which is important when evaluating the expression of differentially spliced proteins.
However, the method also has drawbacks: judging by the results presented, it is possible to value the activity of only a few genes at the same time, and the sensitivity to the expression of some of them barely reaches 10 percent. Experiments on longer-lived animals than mice will allow us to tell how long readr-RNA lives in cells, and whether CellREADR has a future in evaluating the results of cellular and genomic therapy of human diseases.
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